14C-acetyl) phenacetin N-O-glucuronide in Tris buffer (t-1/2 equals 8.7 hr) decomposed to phenacetin, 2-hydroxyphenacetin glucuronide, acetaminophen, and acetamide. In the presence of protein, covalent binding occurred and only acetaminophen and acetamide were decreased. It was thus postulated that acetaminophen, acetamide and covalent binding were formed from N-acetylimidoquinone. In phosphate buffer the N-O-glucuronide is also converted to 3-hydroxyphenacetin phosphate at the expense of covalent binding, acetaminophen and acetamide. In the absence of protein, phosphate completely inhibits acetamide formation but only partially inhibits acetaminophen formation. In the presence of protein, however, phosphate completely inhibits acetaminophen and acetamide and only partially inhibits covalent binding. These data indicate that three reactive intermediates are formed - an ethylated metabolite which reacts with phosphate and two deethylated metabolites which can bind to protein. Only one reactive intermediate which binds to protein can be inhibited by phosphate. The other reactive metabolite which cannot be inhibited by phosphate does not lead to acetamide but can be reduced to acetaminophen.